Labelled factor XIIIa substrates

ABSTRACT

A complex of a radiometal or paramagnetic metal ion with a metal chelating agent such as a diaminedioxime has attached thereto a substituent of formula —(Y) m —A—NHR, can function as a substrate for the fibrin-stabilizing Factor XIIIa. The complex is useful for the diagnosis or therapy of thrombosis, embolism, atherosclerosis, inflammation or cancer.

This application is a 371 of PCT/GB98/00157 filed Mar. 19, 1998.

The present invention relates to a class of compounds useful in thediagnosis of sites of thrombosis, embolism or infection, pharmaceuticalformulations containing them, their use in the diagnosis of disease andmethods for their preparation.

Prior approaches to thrombus imaging radiopharmaceuticals includeradiolabelled fibrinogen or plasminogen; radiolabelled fragment E, ofhuman fibrin; radiolabelled plasminogen activators such as tissueplasminogen activator (t-PA) and labelled anti-fibrin antibodies.Methods based on the detection of sites of platelet accumulation such asthe administration of radiolabelled platelets (e.g. using ¹¹¹In oxine)or radiolabelled anti-platelet antibodies have also been described. Morerecent efforts have focused on radiolabelled peptides or polypeptidessuch as the cell adhesion motif RGD (where R, G and D are the standardabbreviations for the amino acids arginine, glycine and aspartic acidrespectively); platelet factor 4 or fragments thereof or anticoagulantpeptides such as disintegrins.

Factor XIII is a plasma glycoprotein which is present in blood andcertain tissues in a catalytically inactive (or zymogen) form. FactorXIII is transformed into its active form Factor XIIIa by thrombin in thepresence of calcium ions. Factor XIIIa is also known as plasmatransglutaminase, fibrinoligase or fibrin-stabilizing factor. The finalstep in the formation of a blood clot is the covalent crosslinking ofthe fibrin which is formed by the proteolytic cleavage of fibrinogen bythrombin. Fibrin molecules align and the enzyme Factor XIIIa catalysescovalent crosslinking of the NH₂ and CO₂NH₂ groups of lysyl andglutaminyl residues respectively giving structural rigidity to the bloodclot. The crosslinking stabilises the fibrin clot structure and confersresistance to fibrinolysis. The crosslink formation is an importantfacet of normal blood coagulation and wound healing as well aspathological conditions such as thrombosis. It may also be implicated inatherosclerosis and tumour growth and metastasis. WO 91/16931 disclosesthat radiolabelled analogues of Factor XIII (in which the active sitehas been inactivated by amino acid substitution) are useful as thrombusimaging radiopharmaceuticals.

Factor XIIIa is also known to catalyse the incorporation of lowmolecular weight amines into the γ-glutamine sites of proteins. Thussuch low molecular weight amines function as competitive inhibitors ofthe Factor XIIIa-induced glutaminyl crosslinking of proteins. A range ofsynthetic amines have been described which are competitive inhibitors ofthe uptake of labelled putrescine (1,4-butanediamine) intoN,N′-dimethylcasein catalysed by pig liver transglutaminase [L. Lorandet al., Biochem., 18, 1756(1979)].

The possible use of radiolabelled diamines of formula H₂N(CH₂)_(n)NHR*(n and R* undefined) as potential clot imaging agents was disclosed byRhodes et al (Chapter 54, p.521 in “Radiopharmaceuticals”, G.Subramanian, B. A. Rhodes, J. F. Cooper & V. J. Sodd [Eds], Society ofNuclear Medicine Inc., 1975). They envisaged that a radiolabelled aminewhich was an inhibitor of the crosslinking of fibrin could form asubstrate for Factor XIIIa and hence become attached to the fibrin ofblood clots. U.S. Pat. No. 4,406,075 (Mallinckrodt) disclosesradiolabelled aliphatic amines for blood clot imaging of formula:

Y(CH₂)₂—X—(CH₂)₂NH₂

where X is O, S, Se*, Te* or lower alkylene.

When X is Se* or Te*, Y is a hydrocarbylamino group, and

when X is O, S or lower alkylene, Y is a radioiodinated hydrocarbylaminogroup (or salt thereof).

(* denotes a radioactive atom).

WO 89/00051 (Cytrx Biopool Ltd.) claims a method for targeting fibrindeposits using a labelled compound which is covalently bound to fibrinby Factor XIIIa. The fibrin binding compound is stated to be “anypeptide that is a substrate for the blood enzyme commonly known asFactor XIIIa”.

It has now been discovered that metal complexes with suitable pendantfunctional groups can also function as substrates for the enzyme FactorXIIIa. Since Factor XIIIa is only released at pathological sites from aninactive precursor, targeting this enzyme provides a means of targetinga diagnostic imaging agent to the site of Factor XIIIa release.

The present invention provides in one aspect a metal complexing agenthaving attached thereto at least one substituent of formula

—(Y)_(m)—A—NHR,

where:

Y is the same or different at different locations within the moleculeand is independently chosen from: an A group, a C₄₋₈ cycloheteroalkylenegroup, a C₄₋₈ cycloalkylene group, a C₅₋₁₂ arylene group, a C₃₋₁₂heteroarylene group, or a polyalkyleneglycol, polyactic acid orpolyglycolic acid moiety,

m is an integer of value 0-20,

A is a 3-10 atom chain of units selected from —CR₂—, —CR═CR—, —C≡C—,—NRCO—, —CONR—, —SO₂NR—, —NRSO₂—, or —CR₂ZCR₂— where Z is —CH₂—, O, S,Se or —NR—,

R is the same or different at different locations within the moleculeand is independently chosen from H, C₁₋₄ alkyl, C₁₋₄ alkenyl, C₁₋₄alkynyl, C₁₋₄alkoxyalkyl or C₁₋₄ hydroxyalkyl, with the proviso that thecomplexing agent does not also have attached thereto a hypoxialocalising moiety.

The invention also provides a metal complex of one or more radiometal orparamagnetic metal ions with the metal complexing agent as defined; bothas a new compound per se and also for use in the diagnosis or therapy ofthrombosis, embolism, atherosclerosis, inflammation or cancer.

Preferably the metal complexing agent is a metal chelating agent, forexample a diaminedioxime. In preferred substituents, A is

—NHCO(CH₂)₂Z(CH₂)₂—, or

—SO₂NH(CH₂)₂Z(CH₂)₂—, or

—(CH₂)₂Z(CH₂)₂—.

Preferably Z is CH₂. Particularly preferred substituents have theformula

—(Y)_(b)—Ar—SO₂NH(CH₂)₅NH₂

where b is an integer of value 0 to 19 and Ar is an arylene orheteroarylene group.

The complexing agents of the present invention preferably only have asingle type of targeting molecule attached, i.e. the —(Y)_(m)—A—NHRsubstituent. Other substituents on the complexing agent may be present,but the —(Y)_(m)—A—NHR substituent is the one which is expected to beprimarily responsible for the biolocalisation properties. The—(Y)_(m)—A—NHR substituent may be attached to either the backbone whichconnects metal donor atoms of a metal complexing or chelating agent, orto a metal donor atom of the metal complexing or chelating agent.

When Y includes a biocompatible, hydrophilic polymer such as apolyalkyleneglycol, polylactic acid or polyglycolic acid, this polymericlinking group may be useful to prolong the residence time of the metalcomplex in the bloodstream, ie. to slow down the rate of clearance fromthe blood following administration. The hydrophilic polymer ispreferably a polyalkyleneglycol, most preferably polyethyleneglycol(PEG). The hydrophilic polymer preferably has a molecular weight of2,000 to 20,000 Daltons.

The metal complex of the present invention may contain one or more metalions which may be the same or different. Thus in some circumstancespolynuclear complexes may have advantageous properties such as certainmetal clusters which have superparamagnetic properties and are henceparticularly useful as MRI contrast agents. Preferred metal complexes ofthe present invention involve only a single metal ion. When the metal ofthe metal complex is a radiometal, it can be either a positron emitter(such as ⁶⁸Ga or ⁶⁴Cu) or a γ-emitter such as ^(99m)Tc, ¹¹¹In, ^(113m)Inor ⁶⁷Ga. Suitable metal ions for use in MRI are paramagnetic metal ionssuch as gadolinium(III) or manganese(II). Most preferred radiometals fordiagnostic imaging are γ-emitters, especially ^(99m)Tc. Metal complexesof certain radionuclides may be useful as radiopharmaceuticals for theradiotherapy of various diseases such as cancer or the treatment ofthrombosis or restenosis. Useful radioisotopes for such radiotherapeuticapplications include: ⁹⁰Y, ⁸⁹Sr, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁶⁹Er, ¹⁵³Sm and¹⁹⁸Au. Whichever metal complex is chosen, it is strongly preferred thatit is bound to the Factor XIIIa substrate in such a way that it does notundergo facile metabolism in blood with the result that the metalcomplex is cleaved from the Factor XIIIa substrate before the labelledFactor XIIIa substrate reaches the desired in vivo site to be imaged.The Factor XIIIa substrate is therefore preferably covalently bound tothe metal complexes of the present invention.

The metal ions of the present invention are complexed using a metalcomplexing agent or more preferably a metal chelating agent. Thechelating agents comprise 2-10 metal donor atoms covalently linkedtogether by a non-coordinating backbone. Preferred chelating agents have4-8 metal donor atoms and have the metal donor atoms in either an openchain or macrocyclic arrangement or combinations thereof. Most preferredchelating agents have 4-6 metal donor atoms and form 5- or 6-memberedchelate rings when coordinated to the metal centre. Such polydentateand/or macrocyclic chelating agents form stable metal complexes whichcan survive challenge by endogenous competing ligands for the metal invivo such as transferrin or plasma proteins. Alternatively it ispossible to use monodentate complexing agents that form strong stablecomplexes with the desired metal ions. Examples of known complexingagents of this kind, which are particularly suitable for use with^(99m)Tc, are hydrazines, phosphines, arsines and isonitriles. Unlikechelating agents, complexing agents do not necessarily occupy all theco-ordination centres of the metal ion.

The metal complex should also preferably be of low lipophilicity (sincehigh lipophilicity is often related to non-specific uptake), and exhibitlow plasma protein binding (PPB) since plasma-bound label againcontributes to undesirable high, non-specific blood background for theimaging agent.

Examples of suitable chelating agents are diaminedioximes (U.S. Pat. No.4,615,876) or such ligands incorporating amide donors (WO 94/08949); thetetradentate ligands of WO 94/22816; N₂S₂ diaminedithiols,diamidedithiols or amideaminedithiols; N₃S thioltriamides; N₄ ligandssuch as tetraamines, macrocyclic amine or amide ligands such as cyclam,oxocyclam (which forms a neutral technetium complex) or dioxocyclam; ordithiosemicarbazones. The above described ligands are particularlysuitable for technetium, but are useful for other metals also. Othersuitable ligands are described in Sandoz WO 91/01144, which includesligands which are particularly suitable for indium, yttrium andgadolinium, especially macrocyclic aminocarboxylate and aminophosphonicacid ligands. Ligands which form non-ionic (i.e. neutral) metalcomplexes of gadolinium are known and are described in U.S. Pat. No.4,885,363. The ligand may also comprise a short sequence of amino acidssuch as the Cys/amino acid/Cys tripeptide of WO 92/13572 or the peptideligands described in EP 0719790 A2

It is well known to prepare chelating agents which have attached theretoa functional group (“bifunctional chelates”). Functional groups whichhave been attached to chelating agents include: amine, carboxylic acid,cyanate, thiocyanate, maleimide and active ester such asN-hydroxysuccinimide. Examples of chelate-amine conjugates fordiaminedioxime ligands are given in WO 95/19187. The ligands of thepresent invention can be prepared by reaction of a bifunctional compoundwhich contains both an amine group (preferably protected by use ofsuitable protecting groups known to those skilled in the art), and areactive group such as a sulphonyl chloride, acid chloride, active esteror an alkyl/benzyl halide. The reactive group can then be coupled toeither the pendant amine group of a bifunctional chelate, or used toderivatise one or more of the amine donor atoms of a N-containingligand. Alternatively, a mono-protected diamine could be reacted with abifunctional chelate with a pendant active ester or carboxyl group togive the protected amine group linked to the ligand system via an amidebond. In both synthetic routes outlined above, the resultingligand-protected amine conjugate is then deprotected under suitableconditions to give the desired amine-functionalised ligand.

The metal complexes of the present invention may be prepared by reactinga solution of the metal in the appropriate oxidation state with theligand at the appropriate pH. The solution may preferably contain aligand which complexes weakly to the metal (such as chloride, gluconateor citrate) i.e. the metal complex is prepared by ligand exchange ortranschelation. Such conditions are useful to suppress undesirable sidereactions such as hydrolysis of the metal ion. When the metal ion is^(99m)Tc, the usual starting material is sodium pertechnetate from a⁹⁹Mo generator. Technetium is present in ^(99m)Tc-pertechnetate in theTc(VII) oxidation state, which is relatively unreactive. The preparationof technetium complexes of lower oxidation state Tc(I) to Tc(V)therefore usually requires the addition of a suitable reducing agentsuch as stannous ion to facilitate complexation. Further suitablereductants are described below.

Thus the present invention relates to diagnostic agents for imagingsites in the mammalian body where the enzyme Factor XIIIa isup-regulated and fibrin is deposited. The present agents areparticularly useful for the diagnostic imaging of the human body. Theagents comprise substrates for the enzyme Factor XIIIa which arelabelled with a metal complex suitable for external imaging such as aradiometal (for scintigraphy) or a paramagnetic metal ion (for MRI). Themetal complex of the present invention has a pendant amino functionalgroup which is available for covalent linking to protein glutamylcarboxamide groups by the enzyme Factor XIIIa. The intimate relationshipof fibrin and Factor XIIIa highlights the potential use of the agents ofthe present invention for the diagnosis of disease states where there isboth fibrin deposition or accumulation and up-regulation of FactorXIIIa. Increased fibrin deposition is known to be characteristic ofdiseases such as thrombosis, atherosclerosis, fibrotic liver, anddisseminated intravascular coagulation. Fibrin is also deposited atsites of tissue inflammation associated with many disease processes,such as infection, autoimmune disease or cancer. Factor XIIIa and tissuetransglutaminase are up regulated during known physiological conditions.During apoptosis and generation of new matrix protein structureselevated levels of the enzymes are seen. The present agents may thusalso be used for the detection of apoptosis and diseases states such asarthritis where increased matrix protein deposition occurs. Since FactorXIIIa is up-regulated at the site of interest in vivo (i.e. thrombus,embolism etc.) this provides a localisation mechanism for the metalcomplexes of the present invention. The covalently linked metalcomplexes can then be imaged externally by radionuclide scintigraphy ormagnetic resonance imaging (MRI) hence providing a non-invasive means ofdiagnosing the disease site.

The present invention also relates to kits for the preparation of metalcomplexes linked to Factor XIIIa substrates. The kits are designed togive sterile products suitable for human administration, e.g. viainjection into the bloodstream. Possible embodiments are discussedbelow. When the detectable moiety is ^(99m)Tc, the kit would comprise avial containing the free ligand or chelating agent for the metaltogether with a pharmaceutically acceptable reducing agent such assodium dithionite, sodium bisulphite, ascorbic acid, formamidinesulphinic acid, stannous ion, Fe(II) or Cu(I), preferably a stannoussalt such as stannous chloride or stannous tartrate. Alternatively, thekit could contain a metal complex which, upon addition of the radiometalor paramagnetic metal, undergoes transmetallation (i.e. ligand exchange)giving the desired product. For ^(99m)Tc, the kit is preferablylyophilised and is designed to be reconstituted with sterile^(99m)Tc-pertechnetate (TcO₄ ⁻) from a ^(99m)Tc radioisotope generatorto give a solution suitable for human administration without furthermanipulation.

The agents of the present invention may also be provided in a unit doseform ready for human injection and could for example be supplied in apre-filled sterile syringe. When the detectable moiety is a radioactiveisotope such as ^(99m)Tc, the syringe containing the unit dose wouldalso be supplied within a syringe shield (to protect the operator frompotential radioactive dose).

The above kits or pre-filled syringes may optionally contain furtheringredients such as buffers; pharmaceutically acceptable solubilisers(e.g. cyclodextrins or surfactants such as Pluronic, Tween orphospholipids); pharmaceutically acceptable stabilisers or antioxidants(such as ascorbic acid, gentisic acid or para-aminobenzoic acid) orbulking agents for lyophilisation (such as sodium chloride or mannitol).

The following Examples illustrate the preparation of compounds of thepresent invention and their use in imaging. The syntheses of particularcompounds of the present invention are given in Examples 1-20, and theirradiolabelling with ^(99m)Tc in Examples 21-23. Evidence for uptake inblood clots in vitro and in vivo is given in Examples 25, 26, 28 withnormal rat biodistribution of the radiolabelled compounds reported inExample 27. The in vivo results of Example 27 indicate a rapid clearanceof the compounds from the blood. The kinetics of disappearance ofradioactivity from key background organs (e.g. lung, heart, muscle)followed a pattern similar to that of the blood. The analyses of avariety of organs did not show any specific accumulation, with theexception of those compounds that excreted primarily by thehepatobiliary system (HBS) which tended to accumulate in the liver. Therapid background clearance and lack of organ accumulation of thecompounds of the present invention are important characteristics fordiagnostic imaging agents since the area of interest (e.g. thrombus) istherefore more clearly delineated. The percentage of the injected dose(%ID) found in the blood at 60 minutes post injection for the compoundsvaries between 0.3-2.5% with most of the injected dose excreted within 4h post-injection. The majority of compounds have the HBS as the mainroute of excretion with 70-90%ID excreted by this route within 4 h butin several compounds (Compound 17, Compound 24, Compound 25) the urinarysystem is the major excretory route with 50-90%ID being excreted within4 h. The biological half-life of these compounds does vary, but for allof them it is very short. The relatively short half-life of thesecompounds and lack of accumulation at key organs is important for thepossible use in diagnostic imaging of thrombi.

The compounds of the present invention exhibit clot uptake in an animalmodel (Example 28). Higher clot uptake (%ID/g 0.2-0.3%) is seen forthose tracers which have a slower clearance rate. The clot/backgroundratios are, in general, favourable for imaging (i.e. >1) but for thosecompounds excreted via the HBS the clot/liver ratios are poorer.

Com- Ligand R pound

H  2

 3

 4

 5

 6

 8

 9

10

11

13

14

16

17

18

19

20

23

24

25

EXPERIMENTAL Example 1 Synthesis of3,3,6,9,9-pentamethyl-6-(4-(N-(5-aminopentyl)amidosulphonyl)benzamidomethyl)-4,8-diazaundecane-2,10-dionedioxime, trifluoroacetate salt (Compound 3).

(a): Synthesis of 4-(N-(5-N′-^(t)butoxycarbonylaminopentyl)amidosulphonyl)benzoic acid (Compound 1)

To a solution of mono-N-^(t)butoxycarbonyl-1,5-diaminopentane (543 mg,2.69 mmol; Fluka Chemicals) in dichloromethane (10 ml) was addedtriethylamine (748 ml, 5.37 mmol, 2 eq). A slurry of4-chlorosulphonylbenzoic acid (592 mg, 2.69 mmol, 1 eq) indichloromethane (3 ml) was added and the flask rinsed with moredichloromethane (5 ml). The resulting yellow solution was stirred atroom temperature for 19 hours. The solvent was removed in vacuo and theresidue was taken up in 10%aq HCl. It was then extracted with threeportions of EtOAc. The combined organic layers were dried (Na₂SO₄),filtered and evaporated. The residue was then recrystallised from ethylacetate/petrol to afford the title compound as a buff powder (667 mg,64%).

δH (270 MHz, CD₃OD) 1.16-1.5 (15H, m, —C(CH ₃)₃, —(CH ₂)₃—), 2.86 (2H,t, J 8.44 Hz, —CH ₂NHBoc), 2.94 (2H, t, J 8.44 Hz, —SO₂NHCH ₂—), 7.92(2H, d, J 8.44 Hz, aromatic protons), 8.19 (2H, d, J 8.44 Hz, aromaticprotons).

(b): Synthesis of Compound 3

To a solution of Compound 1 (202 mg, 0.52 mmol, 1.1 eq) and Py-BOP (272mg, 0.52 mmol, 1.1 eq) in DMF (5 ml) under N₂ was added DIPEA (91 ml,0.52 mmol, 1.1 eq).The mixture was stirred for 2 hours at roomtemperature, when HPLC showed the formation of a new species withretention time 14.7 mins (System A). A solution of Compound 2(6-aminomethyl-3,3,6,9,9-pentamethyl-4,8-diazaundecane-2,10-dionedioxime, 150 mg, 0.48 mmol; prepared as described in WO 95/19187) in DMF(2 ml ) was added and the mixture was stirred for 3 hours at roomtemperature. After this time HPLC showed the disappearance of the peakat 14.7 mins, and a further new species at 10.1 mins. The reactionmixture was poured into a saturated solution of sodium bicarbonate andextracted with three portions of ethyl acetate. The combined organiclayers were dried (Na₂SO₄), filtered and evaporated. The crude materialwas taken up in ethyl acetate (20 ml) from which a 10 ml aliquot of wastaken and evaporated to dryness. It was then treated with TFA (3 ml) for3.5 hours. After this time had elapsed, HPLC (System A) showed that allthe starting material had been consumed. The mixture was evaporated todryness and purified by RP-HPLC (System B).

Mass Spec. Analysis (FAB+)

Theoretical molecular weight: 583.6

Experimental molecular weight [M+H]+584.3

Example 2 Synthesis of3,3,6,9,9-pentamethyl-6-N-(6-aminohexoyl)aminomethyl-4,8-diazaundecane-2,10-dionedioxime (Compound 4)

Synthesis is as described for Compound 3 but, to a solution of Compound2 in DMF (2 ml) were successively added BOC-6-aminohexanoic acid, Py-BOPand DIPEA. The title compound was obtained by TFA deprotection andRP-HPLC as described for Compound 3.

Mass Spec. Analysis (FAB+)

Theoretical molecular weight: 428.6

Experimental molecular weight [M+H]+429.4

Example 4 Synthesis of3,3,6,9,9-pentamethyl-6-N-[N-(6-aminohexanoyl)-D-phenylalanyl]aminomethyl4,8-diazaundecane-2,10-dionedioxime (Compound 5)

BOC-6-aminohexanoic acid (2.14 g, 9.25 mmol) was coupled toD-phenylalanine benzyl ester hydrochloride (2.7 g, 9.25 mmol) withPy-BOP in the presence of DIPEA, and the resulting product washydrogenated in 95% ethanol at atmospheric pressure in the presence of10% palladium over charcoal to affordBOC-6-aminohexanoyl-D-phenylalanine (3.35 g, 85%). The title compoundwas obtained by coupling BOC-6-aminohexanoyl-D-phenylalanine to Compound2, deprotection and RP-HPLC as described for Compound 3.

Mass Spec. Analysis (FAB+)

Theoretical molecular weight: 575.8

Experimental molecular weight [M+H]+575.9

Example 5 Synthesis of3,3,6,9,9-pentamethyl-6-N-[4-N-(6-aminohexanoyl)aminobenzoyl]aminomethyl-4,8-diazaundecane-2,10-dionedioxime (Compound 6)

BOC-6-aminohexanoic acid was coupled to 4-aminobenzoic acid benzyl esterhydrochloride through a symmetrical anhydride in the presence of DIPEAand a catalytic amount of DMAP, and the resulting product washydrogenated in 95% ethanol at atmospheric pressure in the presence of10% palladium over charcoal to afford BOC-6-aminohexanoyl-4-aminobenzoicacid. The title compound was obtained by couplingBOC-6-aminohexanoyl-4-aminobenzoic acid to Compound 2, TFA deprotectionand RP-HPLC purification as described for Compound 3.

Mass Spec. Analysis (FAB+)

Theoretical molecular weight: 547.7

Experimental molecular weight [M+H]+547.8

Example 6 Synthesis of3,3,6,9,9-pentamethyl-6-N[4-N-{(4-N-(5-aminopentyl)amidosulphonyl)benzoyl}aminomethylcyclohexylcarbonyl]aminomethyl-4,8-diazaundecane-2,10-dionedioxime (Compound 8)

(a): Synthesis of4-(N-(5-N′-^(t)butoxycarbonylaminopentyl)amidosulphonyl)benzamidomethyl-6-cyclohexanecarboxylic acid (Compound 7)

To a solution of trans4-(aminomethyl)cyclohexane carboxylic acid (1 g,6.36 mmol) in 4M NaOH (15 ml) at 0° C. was added BOC anhydride (1.46 ml,6.36 mmol, 1 eq). The mixture was stirred at 0° C. and allowed to warmto room temperature over the course of 24 hours. A white precipitate wasformed as the reaction progressed, which redissolved with time. After 24hours, the mixture was acidified to pH3 with 10% HCl. At pH6 a lot ofwhite solid was observed. This solid was extracted into EtOAc, andacidification of the aqueous phase then continued. When the mixturereached pH3, it was extracted with three portions of EtOAc. The combinedorganic extracts were dried (Na₂SO₄), filtered and evaporated to affordthe desired product (1.384 g, 85%), which was used without furtherpurification.

To a solution of trans-4-(N-BOC-aminomethyl)cyclohexane carboxylic acid(1.359 g, 5.28 mmol) in dichloromethane (15 ml), was added DMAP(catalytic amount) and benzyl alcohol (550 ml, 5.28 mmol, 1 eq). DCC (1Msoln. in CH2Cl2, 5.28 ml, 5.28 mml, 1 eq) was added and the mixture wasstirred at room temperature for 3 hours. The reaction mixture was thenfiltered and the filtrate evaporated. The residue was purified by flashcolumn chromatography (eluent 3:1 petrol:EtOAc) to afford the desiredproduct as a yellow oil which solidified to give an off-white solid onstanding (1.742 g, 95%).

To a solution of trans-4-(N-BOC-aminomethyl)cyclohexane carboxylic acid,benzyl ester (1.401 g, 4.03 mmol) in dichloromethane (5 ml) was addedTFA (10 ml). The solution was stirred at room temperature for 10minutes. The solution was evaporated to dryness affording the product asa yellow oil (1.44 g, 99%).

To a solution of trans-4-(aminomethyl)cyclohexane carboxylic acid,benzyl ester (0.5 g, 1.38 mmol) in dry DMF (2 ml) under N₂ was addedCompound 1 (533 mg, 1.38 mmol, 1 eq), HBTU (523 mg, 1.38 mmol, 1 eq) andDIPEA (1.7 ml, 9.76 mmol, 7 eq). The reaction mixture was stirred atroom temperature for 15 hours. The mixture was diluted withdichloromethane (20 ml) and extracted with three portions of 10% citricacid solution. The organic phase was then washed twice with sat. NaHCO₃and then with brine. The organic layer was dried (Na₂SO₄), filtered andevaporated. The residue was purified by flash column chromatography(eluent 7:3 EtOAc:petrol) to afford the desired product as an off-whitefoam (0.554 g, 65%). To a solution of this foam (437 mg, 7.09×10⁻⁴ mol)in ethanol (15 ml) was added 10% palladium over charcoal (440 mg) andcyclohexene (720 ml, 7.09 mmol, 10 eq). The mixture was heated to refluxtemperature for 4.5 hours, filtered and then evaporated to dryness toafford Compound 7 as a white solid (375 mg, 100%). This solid was usedwithout further purification.

δH (CDCl₃, 300 MHz): 7.88 (4H, s, aromatic), 6.667 (1H, br.s,cyclohexyl-CH₂NH), 4.87 (1H, t, J 9.0 Hz, SO₂NH), 4.54(1H, br.s, NHBoc),3.28 (2H, t, J 9.0 Hz, SO₂NHCH ₂), 2.93-3.03 (4H, m, CH ₂NH, CH ₂NHBoc),2.21-2.33 (1H, m, CHCO₂H), 1.85-2.06 (4H, m, 2×ringCH₂), 0.96-1.66 (20H,m, 2×ring CH₂, ring CH, BOC —(CH₂)₃—).

(b): Synthesis of Compound 8

Compound 7 (150 mg, 2.85×10⁻⁴ mol), Compound 2 (90 mg, 2.85×10⁻⁴ mol, 1eq) and HBTU (108 mg, 2.85×10⁻⁴ mol, 1 eq) were dissolved in DMF (2 ml)under N₂. DIPEA (250 ml, 1.43 mmol, 5 eq) was added and the reactionmixture was stirred at room temperature for 5 hours and followed byRP-HPLC (System D). The crude reaction mixture was purified by RP-HPLC(System E) to afford the desired protected product (142 mg, 63%). Thetitle compound was obtained by TFA deprotection as described forCompound 2. The crude product was purified by RP-HPLC (System F) toafford the desired product as a white solid (8 mg, 23%).

Mass Spec. Analysis (FAB+)

Theoretical molecular weight: 722.0

Experimental molecular weight [M+H]+723.0

Example 7 Synthesis of3,3,6,9,9-pentamethyl-6-(4-(N-[(S-aminoethyl)thioethyl]amidosulphonyl)benzamidomethyl)-4,8-diazaundecane-2,10-dionedioxime (Compound 9)

To a solution of thiacadaverine (0.5 g, 4.16 mmol) in dichloromethane (5ml) at −78° C. under N₂ was added slowly a solution of BOC anhydride(478 ml, 2.08 mmol, 0.5 eq) in dichloromethane (1 ml). The reactionmixture was stirred at −78° C. for 5 hours, then allowed to warmgradually to room temperature for 18 hours. During this time, thereaction mixture turned opaque. The solvent was removed by evaporation,and the white residue was purified by flash column chromatography(eluent 80:20:1 CH₂Cl₂:MeOH:NH₃) to afford the product as a yellow oil(388 mg, 85%). To mono-N-Boc-thiacadaverine (369 mg, 1.68 mmol) indichloromethane (5 ml) was added triethylamine (470 ml, 3.36 mmol, 2 eq)and a slurry of 4-chlorosulphonylbenzoic acid in dichloromethane (2 ml)and reacted as described for Compound 1(429 mg, 63%). The title compoundwas obtained as described for Compound 8 by coupling the above acid (150mg, 3.71×10⁻⁴ mol), Compound 2 (117 mg, 3.71×10⁻⁴ mol, 1 eq) and HBTU(141 mg, 3.71×10⁻⁴ mol, 1 eq) in DMF (2 ml), with DIPEA (330 ml, 1.85mmol, 5 eq). TFA deprotection and RP-HPLC (System F) purification wereas described for Compound 3 (27 mg, 52%).

Mass Spec. Analysis (FAB+)

Theoretical molecular weight: 601.0

Experimental molecular weight [M+H]+602.0

Example 8 Synthesis of3,3,6,9,9-pentamethyl-6-N-[N-(4-N-(5-aminopentyl)amidosulphonyl)benzoyl-D-serine-D-serine-D-serine]aminomethyl4,8-diazaundecane-2,10-dionedioxime (Compound 10)

4-(BOC-5-aminopentyl)amidosulphonylbenzoyl-D-Ser(tBu)l-D-Ser(tBu)l-D-Ser(tBu)-OHwas assembled on a 2-chlorotrityl resin by anchoring Fmoc-D-Ser(tBu) tothe resin, and by successive deprotections/couplings cycles (asdescribed in P. Lloyd-Williams, F. Albericio and E. Girald; ChemicalApproaches to the Synthesis of Peptides and Proteins, CRC Press, 1997)with Fmoc-D-Ser(tBu), Fmoc-D-ser(tBu), Fmoc-D-ser(tBu) and Compound 1.The protected compound was obtained by 1% TFA in dichloromethanecleavage. The title compound was obtained by coupling with Py-BOP of theprotected intermediate to Compound 2, TFA deprotection and RP-HPLCpurification as described for Compound 3.

Mass Spec. Analysis (FAB+)

Theoretical molecular weight: 845.0

Experimental molecular weight [M+H]+844.7

Example 9 Synthesis of3,3,6,9,9-pentamethyl-6-N-[4-N{4-(N-(5-aminopentyl)amidosulphonyl)benzoyl}aminomethylpiperazinylcarbonyl]aminomethyl-4,8-diazaundecane-2,10-dionedioxime (Compound 11)

The pseudo-protected peptide was obtained as in Example 8. The titlecompound was obtained by coupling to Compound 2, TFA deprotection andRP-HPLC purification as described for compound 3.

Mass Spec. Analysis (FAB+)

Theoretical molecular weight: 709.9

Experimental molecular weight [M+H]+709.9

Example 10 Synthesis of3,3,6,9,9-pentamethyl-6-N{4-(N-(5-aminopentyl)amidosulphonyl)benzoyl}amidododecanoyl]aminomethyl-4,8-diazundecane-2,10-dionedioxime (Compound 13)

(a): Synthesis of4-(N-(5-N′-^(t)butoxycarbonylaminopentyl)amidosulphonyl) benzoylamidododecanoic acid (Compound 12)

12-aminododecanoic acid (1 g, 4.64 mmol) was BOC protected as describedin Example 7. (0.467 g, 35%). The benzyl ester of the above was formedas described in Example 6 to afford the desired compound as a viscousoil (0.821 g). To a solution of the N-BOC-12-aminododecanoic acid,benzyl ester (821 mg) in dichloromethane (5 ml) was added TFA (5 ml)dropwise with stirring. The solvents were then removed in vacuo toafford the desired product. This product was used without any furtherpurification. To a solution of the resulting crude product (1 g) in DMF(3 ml) under N₂ was added Compound 1 (1.27 g, 3.29 mmol), HBTU (1.24 g,3.27 mmol) and DIPEA (2 ml, 11.48 mmol). The reaction was stirred atroom temperature until TLC (3:7, petrol:EtOAc) indicated that thereaction was complete. The reaction mixture was diluted withdichloromethane, then washed with three portions of 10% citric acidsolution. The organic phase was washed with saturated sodium bicarbonatesolution and finally with brine. The organic layer was dried (Na₂SO₄),filtered and evaporated. The crude residue was subjected to flash columnchromatography (3:7, petrol:EtOAc) to afford the desired compound as ayellow solid (1.433 g, 66%).

To a solution of the benzyl protected compound (1.4 g, 2.07 mmol) inethanol (20 ml) was added 10% palladium over charcoal (1.4 g) andcyclohexene (1 ml, 9.87 mmol). The reaction mixture was heated at refluxtemperature until TLC (3:7, petrol:EtOAc) indicated that the reactionwas complete. The reaction mixture was then cooled and filtered. Thefiltrate was concentrated in vacuo to afford an oily yellow residuewhich was purified by RP-HPLC (System G) to afford the desired product(80 mg).

Mass Spec. Analysis (ES−)

Theoretical molecular weight: 583.0

Experimental molecular weight [M−H]+582.0

(b): Synthesis of Compound 13

To a solution of Compound 12 (70 mg, 1.2×10⁻⁴ mol), Compound 2 (50 mg,1.58×10⁻⁴ mol, 1.3 eq) and HBTU (59 mg, 1.56×10⁻⁴ mol, 1.3 eq) in dryDMF (4 ml) under N₂ was added DIPEA (135 mi, 9.13×10⁻⁴ mol, 6.5 eq). Thereaction mixture was stirred at room temperature and then purified byRP-HPLC (System G) to afford the intermediate as a white solid (60 mg,57%). The title compound was obtained by TFA deprotection as describedfor Compound 3 and purified by RP-HPLC (System H) (10 mg, 23%).

Mass Spec. Analysis (ES+)

Theoretical molecular weight: 780.0

Experimental molecular weight [M+H]+781.6

Example 11 Synthesis of3,3,6,9,9-pentamethyl-6-N-[α-N-(4-N-(5-aminopentyl)amidosulphonyl)benzoyl)amino-polyethyleneglycol)-ω-carbonyl]aminomethyl-4,8-diazaundecane-2,10-dionedioxime (Compound 14)

A solution of α-N-(tert-butoxycarbonyl)-poly(ethyleneglycol)amino-ω-succinimidyl carbonate (500 mg, ˜147 mmol) and Compound 2(46.3 mg, 147 mmol) in dry THF (5 ml) was refluxed for 5 hours under N₂.The reaction mixture was reduced in vacuo leading to a white solid whichwas purified by flash chromatography (¹PrOH/NH₃/H₂O, 10:1:1) to give awhite solid (456 mg, 86%). 37% HCl (1.34 ml) was added dropwise to anice cold solution of the above (520 mg, ˜144 mmol) in methanol (3.16ml). The solution was then stirred at room temperature for 4 hours. Thereaction mixture was basified to pH 10 by addition of 4M NaOH (4.18 ml),and the resulting product was isolated by RP-HPLC (System I). To asolution of the above (100 mg, 28.6 mmol) and compound 1 (11 mg, 28.6mmol) in DMF (2 ml) was added HBTU (10.8 mg, 28.6 mmol) and DIPEA (25ml, 143 mmol). The reaction mixture was stirred at room temperature,under N₂, for 22 hours then diluted with dichloromethane (20 ml), washedwith NaHCO₃ (10 ml), water (10 ml), and brine (10 ml). The organic layerwas reduced in vacuo and the residue was purified by RP-HPLC (System J)to give a colourless oil (36.8 mg, 33%). The oil (36.8 mg, ˜9.5 mmol)was dissolved in a solution of 3M HCl in methanol (600 ml) and stirredat room temperature for 5.5 hours before the reaction mixture wasbasified to pH ˜10 by addition of 4M NaOH (550 ml). The title compoundwas obtained by RP-HPLC (System K) as a white gum (29.5 mg, 82%).

Mass Spec. Analysis (MALDI-TOF)

Theoretical molecular weight range: 3000-5000

Experimental molecular weight range [M+H]+3000-5000

Example 12 Synthesis of3,3,11,11-tetramethyl-7-(4-N-(5-aminopentyl)-amidosulphonyl)benzamidoethyl-4,7,10-triazatridecane-2,12-dionedioxime (Compound 16)

(a): Synthesis of3,3,11,11-tetramethyl-7-aminoethyl-4,7,10-triazatridecane-2,12-dionedioxime(Compound 15)

To a solution of tris-(2-aminoethyl)amine (1 ml, 6.68 mmol) inacetonitrile (10 ml) was added sodium bicarbonate (1.12 g, 13.36 mmol, 2eq). A solution of 3-chloro-3-methyl-2-nitrosobutane (1.359 g, 10.02mmol, 1.5 eq) in dry acetonitrile (5 ml) was added slowly. The reactionmixture was left to stir at room temperature for 3 days, and thenfiltered. The residue was washed well with acetonitrile, and thefiltrate evaporated. The crude product was then purified by RP-HPLC(System L) to afford Compound 15 (164 mg, 7%).

δ_(H) (CD₃OD, 300 MHz): 2.77 (2H, t, J 6Hz, CH ₂NH₂), 2.50-2.58 (10H, m,H₂NCH₂CH ₂N(CH ₂CH ₂NH)₂), 1.85 (6H, s, 2×CH ₃C═N), 1.23 (12H, s, 2×(CH₃)₂CNH).

(b): Synthesis of Compound 16

To a solution of Compound 15 (201 mg, 0.583 mmol) in DMF (2 ml) weresuccessively added Compound 1 (225 mg, 0.583 mmol). Py-BOP (258 mg,0.583 mmol) and DIPEA (0.102 ml, 0.583 mmol) and the mixture was stirredfor 2 hours at room temperature. The reaction mixture was diluted withethyl acetate (50 ml), washed with saturated sodium bicarbonate, driedover sodium sulphate and concentrated in vacuo to leave a foamy residue.The above residue was dissolved in a mixture of TFA and water (95/5,v/v, 10 ml) and the solution stirred for 1 hour at room temperature. Thetitle compound precipitated upon addition of diethyl ether (150 ml). Itwas collected and washed with diethyl ether, dried in vacuo and purifiedby RP-HPLC (System A).

Mass Spec. Analysis (ES+)

Theoretical molecular weight: 612.8

Experimental molecular weight [M+H]+612.4

Example 13 Synthesis of3,311,11-tetramethyl-7-N-[4-N-(5-aminopentyl)amidosulphonyl)benzoyl)aminomethylcyclohexylcarbonyl]aminoethyl-4,7,10-triazatridecane-2,12-dione dioxime (Compound17)

To a solution of Compound 7 (84 mg, 1.6×10⁻⁴ mol), Compound 15 (65 mg,1.89×10⁻⁴ mol) and HBTU (61 mg, 1.6×10⁻⁴ mol, 1 eq) in dry DMF (2 ml)under N₂, was added DIPEA (140 ml, 8.00×10⁻⁴ mol, 5 eq). The reactionwas stirred at room temperature for 3 hours and the crude mixture waspurified by RP-HPLC (System L) to afford the desired product (56 mg,41%). The title compound was obtained by TFA deprotection as describedfor Compound 3 and purified by RP-HPLC (System L) (19 mg, 39%).

Mass Spec. Analysis (ES+)

Theoretical molecular weight: 751.3

Experimental molecular weight [M/2+H ]+376.1

Example 14 Synthesis of 1-N-[4-(N-(5-aminopentyl)-amidosulphonyl)benzoyl-3,7-diazanonyl-1,9-diamine(Compound 18)

To a cold solution (−70° C.) ofN,N′-bis(2-aminoethyl)-1,3-propanediamine (2 g, 12.48 mmol) in anhydrousdichloromethane (20 ml) was added dropwise, under N₂, a solution ofdi-tert-butyl dicarbonate in dichloromethane (4 ml). The reactionmixture was stirred at −70° C. for 0.5 hour and at room temperature for18 hours. The solvent was removed in vacuo to give an oil which waschromatographed over silica gel (CH₂Cl₂/MeOH/NH₃, 80:20:2) yielding themono-protected tetraamine (1.08 g, 33%).

A solution of Compound 1 (170 mg, 0.44 mmol) and N-hydroxysuccinimide(51 mg, 0.44 mmol) in anhydrous tetrahydrofuran (4 ml) was cooled to−10° C., under N₂. A 1M solution of DCC in dichloromethane (440 ml, 0.44mmol) was added dropwise and the reaction mixture was stirred at roomtemperature for 1.5 h. The reaction mixture was then treated with asolution of the mono-protected tetraamine (95.6 mg, 0.37 mmol) inanhydrous THF (4 ml). After stirring at room temperature for 18 hours,the reaction mixture was reduced in vacuo. The white solid obtained waschromatographed over silica gel (CH₂Cl₂/MeOH/NH₃, 90:20:2) to give acolour less oil (120 mg, 52%). A solution of 35% HCl/MeOH 1:1 (2.3 ml)was added dropwise to a solution of the above oil (120 mg, 191 mmol) inmethanol (0.7 ml) over 21 hours. The reaction mixture was stirred for afurther 2 hours and the white solid formed was recovered by filtration,washed three times with methanol (1 ml) and dried in vacuo leading to awhite powder of the hydrochloride salt of the title compound (94.7 mg,86%).

Mass Spec. Analysis (ES+)

Theoretical molecular weight: 429.3

Experimental molecular weight [M/2+H]+215.3

Example 15 Synthesis of1-N-[4-(N-(5-aminopentyl)amidosulphonyl]benzoyl-6-oxo-7,10,14-triazahexadecyl-1,16-diamine(Compound 19)

To a solution of the monoprotected tetraamine as described in Example 14(161 mg, 0.62 mmol) and Z-aminocaproic acid (164 mg, 0.62 mmol) in DMF(3 ml) was added HBTU (235 mg, 0.62 mmol) and DIPEA (539 ml, 3.1 mmol).The reaction mixture was stirred at room temperature, under N₂, for 23hours then diluted with dichloromethane (10 ml), washed with NaHCO₃ (10ml) and water (20 ml). The organic layer was reduced in vacuo and theresidue was chromatographed over silica gel (CH₂Cl₂/MeOH/NH₃, 70:30:2)to give a pale yellow oil (121.7 mg, 39%). To a solution of this oil(116 mg, 0.23 mmol) in anhydrous ethanol (3 ml) was added cyclohexene(232 ml, 2.3 mmol) and 10% palladium over charcoal (116 mg). Thereaction mixture was heated at 60° C. for 2 hours, cooled to roomtemperature and then filtered. The residue was washed with methanol, thefiltrates combined and reduced in vacuo to give a colourless oil (81.2mg, 94.6%). To a solution of the oil (81.2 mg, 0.22 mmol) and Compound 1(84.0 mg, 0.22 mmol) in DMF (3 ml) was added HBTU (82.6 mg, 0.22 mmol)and DIPEA (190 ml, 1.1 mmol). The reaction mixture was stirred at roomtemperature under N₂, for 5 hours then diluted with dichloromethane (15ml), washed with NaHCO₃ (20 ml) and water (2×10 ml). The organic layerwas reduced in vacuo and the residue was chromatographed over silica gel(CH₂Cl₂/MeOH/NH₃, 70:30:2) to give a white gum (73.5 mg, 45%). The titlecompound was obtained by acid deprotection as described for Compound 18,as a pale yellow solid (30.8 mg, 100%).

Mass Spec. Analysis (ES+)

Theoretical molecular weight: 542.3

Experimental molecular weight [M/2+H]+271.8

Example 16 Synthesis of1-(4-(N-(5-aminopentyl)-amidosulphonyl)benzyl)-1,4,8,11-tetraazacyclotetradecane(Compound 20)

To a solution of mono-BOC-1,5-diaminopentane (0.5 ml, 2.40 mmol) indichloromethane (5 ml) was added triethylamine (400 ml, 2.87 mmol, 1.2eq). A solution of bromomethyl benzenesulphonyl chloride (0.648 g, 2.40mmol, 1 eq) in dichloromethane (2 ml) was added dropwise with stirringfor 3 hours. The reaction mixture was washed with water, and the organicphase was dried (Na₂SO₄), filtered and evaporated. The residue waspurified by flash column chromatography (3:2 petrol:EtOAc) to afford thedesired product as an oil (190 mg, 18%). To a solution of cyclam (170mg, 8.49×10⁻⁴ mmol, 7 eq) under N₂ in dry THF (5 ml) and dry ethanol (2ml) was added sodium hydride (8 mg, 2×10⁻⁴ mol, 2 eq). The reactionmixture was stirred for 30 mins, after which a solution ofN-BOC-bromomethyl benzenesulphonyl cadaverine (50 mg, 1.15×10⁻⁴ mol) inTHF (1 ml) was added. The reaction was monitored by TLC (70:30:1CH₂Cl₂:MeOH:NH₃). When the reaction was judged to be complete, thesolvent was removed in vacuo. The residue was taken up indichloromethane, and washed twice with saturated sodium bicarbonatesolution. The organic phase was dried (Na₂SO₄), filtered and evaporated.The crude residue was purified by RP-HPLC (System L) to afford thedesired product (20 mg, 31%). The title compound was obtained by TFAdeprotection as described for Compound 3 and purified by RP-HPLC (SystemL) (7 mg, 43%).

δ_(H) (CDCl₃, 300 MHz): 7.77 (2H, d, J 8.4 Hz, aromatic CH), 7.51 (2H,d, J 8.4 Hz, aromatic CH), 3.61 (2H, s, PhCH ₂), 2.55-2.96 (20H, m,SO₂NHCH ₂, CH ₂NHBoc, 8×cyclam CH ₂N), 1.33-1.54 (6H, m, —(CH ₂)₃—).

Example 17 Synthesis of S-acetyl-mercaptoacetylglycylglycylglycine(Compound 21)

A solution of H-Gly-Gly-Gly-OH (1.28 g, 6.76 mmol),S-acetyl-thioglycolic acid pentafluorophenyl ester (Novabiochem, 2 g,6.76 mmol) and DIPEA (1.14 ml, 6.76 mmol) in a mixture of DMF (25 ml)and water (12 ml), was stirred overnight at room temperature. Thereaction mixture was filtered to remove some remaining H-Gly-Gly-Gly-OH,brought to pH2 with 1M aqueous HCl and evaporated to dryness. Theresidue was triturated with acetone to afford the title compound.

Mass Spec. Analysis (ES+)

Theoretical molecular weight: 305.3

Experimental molecular weight [M+H]+305.8

Example 18 Synthesis ofN-[S-acetyl-mercaptoacatylglycylalycylglycyl)-N′-(4-N-(5-aminopentyl)amidosulphonyl)benzoylpropane-1,3-diamine (Compound 23)

(a): Synthesis of N-4-(N-(5-N′-^(t)butoxycarbonylaminopentyl)sulphonamido)benzoyl)-N′-fluorenylmethoxycarbonylpropane-1,3-diamine (Compound 22)

BOC-1,3-diaminopropane was reacted with Fmoc-OSu in dichloromethane inthe presence of DIPEA, and the resulting product treated with 4M HCl indioxane to afford Fmoc-1,3-diaminopropane hydrochloride in quantitativeyield. To a solution of Fmoc-diaminopropane hydrochloride (1.1 g, 3.3mmol), Compound 1 (1.27 g, 3.3 mmol) and Py-BOP (1.46 g, 3.3 mmol) inDMF (10 ml), was added DIPEA (1.15 ml, 6.6 mmol) and the mixture wasstirred for 4 hours at room temperature. The reaction mixture wasdiluted with EtOAc (200 ml), washed with water, saturated aqueous sodiumhydrogen carbonate, 1M aqueous potassium hydrogen sulphate, brine, andconcentrated under reduced pressure to afford Compound 22 as a whitesolid that was triturated with hexane, collected by filtration and driedin vacuo.

(b): Synthesis of Compound 23

Compound 22 (1 g, 1.5 mmol) was treated with diethylamine (2 ml) in DMFfor 1 hour at room temperature. The solution was concentrated to drynessand the resulting gum triturated in diethyl ether to afford a whitesolid. The solid was coupled to Compound 21 (370 mg) with Py-BOP aspreviously described for Compound 3 to afford a white solid aftertrituration in diethyl ether. The title compound was obtained by TFAdeprotection and RP-HPLC as described for Compound 3.

Mass Spec. Analysis (ES+)

Theoretical molecular weight: 629.8

Experimental molecular weight [M+H]+629.3

Example 19 Synthesis ofN-[6-hydrazinopyridine-3-carbonyl]-N′-(4-N-(5-aminopentyl)amidosulphonyl)benzoylpropane-1,3-diamine (Compound 24)

6-BOC-hydrazinopyridine-3-carboxylic acid N-hydroxysuccinimde ester wasprepared according to Schwartz et al. (U.S. Pat. No. 5,206,370, 1993).It was then coupled to diaminopropane in DMF and purified by silica gelchromatography. The title compound was obtained by coupling the abovewith Compound 1, TFA deprotection and RP-HPLC purification as describedfor Compound 3.

Mass Spec. Analysis (ES+)

Theoretical molecular weight: 477.5

Experimental molecular weight [M+H]+477.3

Example 20 Synthesis ofN,N′-bis-(2-hydroxybenzyl)-2-(4-N-(5-aminopentyl)amidosulphonyl)benzamidomethyl-2-methyl-propane-1,3-diamine(Compound 25)

To a solution of 2-[bis-(aminomethyl)]propylamine (0.5 g, 4.26 mmol) indry dichloromethane (49 ml) under N₂ was added dry triethylamine (0.29ml, 2.08 mmol). The resultant solution was cooled to −78° C., and asolution of BOC anhydride (0.465 g, 2.13 mmol, 0.5 eq) in drydichloromethane (49 ml) was added dropwise over 1 hour. The mixture wasallowed to warm to room temperature over several hours and then stirredovernight at room temperature, during which time a white cloudysuspension formed. 1M NaOH soln. (30 ml) was added, and the organiclayer separated. The aqueous layer was then extracted with threeportions of dichloromethane. The combined organic layers were dried(MgSO₄), filtered and evaporated to afford the desired product as acolourless oil (0.58 g, 63%). To a solution ofmono-N-BOC-2-[bis-(aminomethyl)]propylamine (0.58 g, 2.67 mmol) in dryethanol (27 ml) under argon was added salicylaldehyde (0.57 ml, 5.34mmol, 2 eq). The mixture was heated at reflux temperature for 1 hourduring which time a yellow solution formed. After cooling, the solventwas removed in vacuo to afford the desired product as a yellow oil (0.66g, 58%).

To a solution of the above crude product (0.47 g, 1.11 mmol) in dryethanol (15 ml) was added sodium borohydride (146 mg, 3.86 mml 3.5 eq).The mixture was stirred at room temperature for 2 hours, during whichtime the yellow colour disappeared to give a colourless solution. Water(3 ml) was added, and the ethanol/water was decanted off from the whiteoily solid which was formed. The ethanol was removed in vacuo, and theremaining aqueous phase was extracted with two portions ofdichloromethane. The combined organic layers were dried (MgSO₄),filtered and evaporated to give a colourless oil. The crude oil waspurified by flash column chromatography using a gradient of 70%EtOAc/petrol to 100% EtOAc, affording the desired product as acolourless oil (250 mg, 52%). The oil (105 mg, 2.45×10⁻⁴ mol) wastreated at room temperature for 1 hour with 9:1 TFA:CH₂Cl₂ (5 ml). Thesolvents were removed in vacuo to afford the desired product as an oil(109 mg, 100%).

To a solution of the above oil (109 mg, 2.45×10⁻⁴ mol) and Compound 1(95 mg, 2.45×10⁻⁴ mol, 1 eq) in DMF (2 ml) under N₂ was added HBTU (93mg, 2.45×10⁻⁴ mol, 1 eq) and DIPEA (300 ml, 1.72 mmol, 7 eq). Thereaction mixture was stirred for 24 hours and then diluted withdichloromethane before washing with three portions of 10% citric acid.The organic layer was then washed with two portions of saturated sodiumbicarbonate solution and finally with one portion of brine. The organiclayer was dried (Na₂SO₄), filtered and evaporated to afford a crudeproduct. The crude reaction mixture was taken forward in to the nextstep without purification. The title compound was obtained by TFAdeprotection as described for Compound 3 and purified by RP-HPLC (SystemL) (14 mg).

Example 21 Tc-99 mlabelling of Compounds 2-6, 8-11, 13, 14, 16-20, 25

A 0.1 ml aliquot of the compound dissolved in methanol or H₂O (1 mg/ml;7.5 mg/ml for Compound 14) was transferred to a nitrogen-filled 10 mlglass vial together with deoxygenated saline (0.9% w/v, 1 ml) and 0.035ml aqueous NaOH (0.1M) (for Compound 20 1M NaOH was used). To thissolution was added technetium generator eluate (1 ml, approx. 0.4 GBq)and then aqueous stannous chloride solution (0.1 ml, ca.10 μg). Thelabelling pH was 9.0-10.0 (pH11-12 for Compound 20). Vials wereincubated at ambient laboratory temperature (15-25° C.) for 30 minutesto effect labelling. HPLC purification was performed (System C; System Hfor Compound 14; System N for Compounds 18, 19) to remove unlabelledstarting material and radioactive impurities prior to testing. Afterpurification the organic solvent was removed under vacuum and the samplewas redissolved in about 5 ml 0.1M phosphate buffer pH7.4 to give aworking concentration of 6-9 MBq/ml. Radiochemical purity was assessedbefore use by the thin layer chromatography (TLC) system describedbelow:

i) ITLC SG 2 cm×20 cm eluted with 0.9% w/v saline

ii) Whatman No. 12 cm×20 cm eluted with 50:50 v/v acetonitrile: H₂O

The labelled substrates remain at, or close to, the origin in TLC system(i) and move close to the solvent front in system (ii). When analysed byappropriate detection equipment the radiochemical purity is typically inexcess of 85% labelled compound.

Example 22 Tc-99m labelling of Compound 23

A gluconate kit was reconstituted with technetium generator eluate (5ml, 2 GBq) and allowed to incubate at room temperature for 15 minutes toeffect labelling. An aliquot (0.1 ml) of the compound freshly dissolvedin methanol (5 mg/ml) was transferred to a nitrogen-filled 10 ml glassvial together with 0.025 ml of aqueous NaOH (0.1M) and 2 ml of the^(99m)Tc-gluconate solution. The labelling pH was 9.0. Vials wereincubated at room temperature for 30 minutes to effect labelling.Purification and assessment of radiochemical purity was carried out asfor Example 21.

Example 23 Tc-99 mlabelling of Compound 24

A 0.1 ml aliquot of the compound dissolved in methanol (1 mg/ml) wastransferred to nitrogen-filled 10 ml glass vial together with tricinedissolved in water (0.5 ml, 37.5 mg) and phosphinedynetris(benzenesulphonic acid)tris sodium salt dissolved in water (0.1 ml, 1 mg). Tothis solution was added technetium generator eluate (1 ml, approx 0.4GBq) and then a solution of stannous chloride in 0.1M HCl (0.02 ml, ca 2μg). The labelling pH was 4.5-5.5. Vials were incubated at 60° C. for 30minutes to effect labelling. Purification and assessment ofradiochemical purity was carried out as in Example 21.

Example 24 HPLC Systems

System A

Column Waters C18 150×3.9 mm. Particle size 4 microns

Gradient: Elution Profile 0-100%B in 22 min.

Eluent A: 0.1% aqueous TFA

Eluent B: acetonitrile

Flow Rate: 1 ml/min

System B

Column Waters C18 150×3.9 mm. Particle size 4 microns

Gradient: Elution Profile 0-100%B in 22 min.

Eluent A: 0.1% aqueous TFA

Eluent B: acetonitrile

Flow Rate: 3 ml/min

System C

Column Waters C18 150×3.9 mm. Particle size 4 microns

Gradient: Elution Profile 0-100%B in 22 min.

Eluent A: 0.1% aqueous TFA

Eluent B: 0.1% TFA in acetonitrile

Flow Rate: 1 ml/min

System D

Column Hamilton PRP-1

Gradient: Elution Profile 0-100%B in 20 min.

Eluent A: 0.1% aqueous TEA

Eluent B: 0.1% TEA in acetonitrile

Flow Rate: 1 ml/min

System E

Column Hamilton PRP-1

Gradient: Elution Profile 20-100%B in 15 min.

Eluent A: 0.1% aqueous TEA

Eluent B: 0.1% TEA in acetonitrile

Flow Rate: 3 ml/min

System F

Column Hamilton PRP-1

Gradient: Elution Profile 0-100%B in 20 min.

Eluent A: 0.1% aqueous TEA

Eluent B: 0.1% TEA in acetonitrile

Flow Rate: 3 ml/min

System G

Column Hamilton PLRP-S

Gradient: Elution Profile 0-100%B in 20 min.

Eluent A: 2% aqueous NH₃

Eluent B: acetonitrile

Flow Rate: 6 ml/min

System H

Column Hamilton PRP-1

Gradient: Elution Profile 0-100%B in 20 min.

Eluent A: 2% aqueous NH₃

Eluent B: acetonitrile

Flow Rate: 1 ml/min

System I

Column Hamilton PRP-1

Gradient: Elution Profile 0-65%B in 10 min.

Eluent A: 5% aqueous NH₃

Eluent B: acetonitrile

Flow Rate: 2.5 ml/min

System J

Column: Hamilton PRP-1

Gradient: Elution Profile 0-65%B in 10 min.

Eluent A: 5% aqueous NH₃

Eluent B: acetonitrile

Flow Rate: 2.5 ml/min

System K

Column Hamilton PRP-1

Gradient: Elution Profile 0-65%B in 10 min.

Eluent A: 5% aqueous NH₃

Eluent B: acetonitrile

Flow Rate: 1 ml/min

System L

Column Hamilton PRP-1

Gradient: Elution Profile 0-100%B in 20 min.

Eluent A: 2% aqueous NH₃

Eluent B: acetonitrile

Flow Rate: 3 ml/min

System M

Column Hamilton PRP-1

Gradient: Elution Profile 0-100%B in 22 min.

Eluent A: 5% aqueous NH₃

Eluent B: acetonitrile

Flow Rate: 1 ml/min

System N

Column Waters C18 150×3.9 mm. Particle size 4 microns

Gradient: Elution Profile 0-100%B in 20 min.

Eluent A: 0.1% aqueous TEA

Eluent B: 0.1% TEA in acetonitrile

Flow Rate: 1 ml/min

Example 25 Incorporation into Human Plasma Clots

Incorporation of radiolabelled substrates into fibrin was investigatedby induction of an in vitro human plasma clot in the following manner.To a siliconised 5 ml glass vial was added, (a) 800 μl ofTris(hydroxymethyl)aminomethane buffered saline pH 7.5 containingcalcium chloride (50 mM Tris, 150 mM sodium chloride, 4 mM calciumchloride.), (b) 40 μl of physiological salt solution containing 100units of thrombin per ml, (c ) 400 μl of human plasma containing theradiolabelled substrate at a concentration of typically 10 kBq/ml. Toaid induction of clot a roughened glass rod was added to the reactionvial. Control vials were prepared similarly but with the omission ofthrombin and calcium chloride.

After incubation of the test solution at ambient laboratory temperature(ca. 20° C.) for 60 minutes the reaction was discontinued with theaddition of about 400 μl of a cold solution of 33.5 mMethylenediaminetetra-acetic acid disodium salt. Clots were separatedfrom serum by vacuum filtration onto 0.45 μM nitrocellulose filters(pre-soaked in 1.5% BSA/tris(hydroxymethyl)aminoethane buffered salinepH 7.5 containing 0.1% Tween 20) and washed with about 2×10 ml ofTris(hydroxymethyl)aminomethane buffered saline pH 7.5 containing Tween20 to a final concentration of 0.1%v/v. The proportion of totalradioactivity was calculated by counting in suitable detectionapparatus.

The fraction of radioactivity retained on the filter, after subtractionof the non-specific binding determined from the control, is a measure ofincorporation into the filtered clots.

Example 26 Factor XIIIa Dependency of Incorporation

The radiolabelled substrates may be tested for Factor XIIIa mediatedincorporation into fibrin and its analogues by a modification of themethod of Dvilansky A. et al [Br. J. Haematol., 18 399-410(1970)]. Theassay was changed by replacing the mercaptoethanol with aprotinin at afinal concentration of 2 μg/ml, and dimethylcasein (DMC) was usedinstead of casein. Factor XIIIa specificity of the reaction isdemonstrated by comparing human plasma with Factor XIII deficientplasma.

The modified assay was used to screen the test compounds (1 KBq/ml) anduptake after 60 minutes was determined. Compound activity was expressedas specific uptake by subtracting uptake in Factor XIII deficient plasmafrom normal plasma (see results table). The assay indicated that^(99m)Tc labelled compounds could be produced that had a range of levelsof incorporation. These compounds were shown to be incorporated by aFactor XIII specific manner.

Results

% retained in % retained in % specific plasma clot plasma clot % DMCassay assay specific casein compound (with thrombin) (no thrombin)uptake uptake Tc-Compound 2 2 0.3 1.7 Not done Tc-Compound 3 22 3.0 1940 Tc-Compound 4 3 0.4 2.6 2 Tc-Compound 5 6 1.9 4.1 10 Tc-Compound 6 20.5 1.5 13 Tc-Compound 8 23 4.6 18.4 45 Tc-Compound 9 20 4.5 15.5 29Tc-Compound 10 5 0.3 4.7 6 Tc-Compound 11 16 3.2 12.8 24 Tc-Compound 1435 7.2 27.8 N/A Tc-Compound 16 2 0.8 1.2 1 Tc-Compound 17 17 2.3 14.722.5 Tc-Compound 20 4 0.6 3.4 8 Tc-Compound 23 16 0.8 14.2 24.5Tc-Compound 24 14 0.2 13.8 22.5 Tc-Compound 25 15 1.9 12.9 26 % retainedin plasma clot assay (with thrombin) - % retained in plasma clot assay(no thrombin)

Example 27 Normal Rat Biodistribution

The resolution of a clot image is dependent on the combination of rateof incorporation of the radiopharmaceutical and its blood/tissuesclearance rate. For this reason the biodistribution of several compoundshas been determined in rats. Male Wistar (100-150 g) rats were injectedi.v. with 0.1-0.2 ml of radiolabelled tracer solution (8 MBq/ml) anddissected at different times post-injection. The %ID in each of theselected tissues was measured. Some animals were kept in metabolismcages to be able to determine the %ID excreted in urine and faeces. Thedissection times used for the majority of the agents was 2, 15, 30, 60,240 min. Data are shown as %ID, Mean±SD, (n=3).

Results

Compound 3

2 min 15 min 30 min 60 min 240 min Muscle 23.6 + 0.9  4.87 + 0.6  1.53 +0.71 1.14 + 0.48  0.8 + 0.17 Blood 19.29 + 1.7  1.45 + 0.38 0.59 + 0.070.31 + 0.02 0.11 + 0.07 Kidney 5.55 + 1   2.1 + 0.8 2.09 + 1.87 0.57 +0.12 0.45 + 0.04 Urine 0.06 + 0.02 5.01 + 2.53 14.42 + 3.78  14.66 +2.83  12.31 + 1.93  Lung 1.43 + 0.16 0.23 + 0.12 0.03 + 0.04 0.07 + 0.020.14 + 0.24 Liver 19.34 + 0.8  7.79 + 1.16 1.95 + 0.35 1.54 + 0.291.35 + 0.06 GI Tract 10.1 + 1.2   70 + 3.4 76.5 + 5.6  79.9 + 2.9 83.5 + 2.2  Heart 0.51 + 0.08 n.d. n.d. n.d. n.d. n.d. not detectable.

Compound 4

2 min 30 min 60 min 240 min Muscle 22.6 + 0.8  5.27 + 0.4  2.26 + 0.051.94 + 0.35 Blood 10.33 + 1.2  0.98 + 0.18 0.43 + 0.07 0.21 + 0.08Kidney 8.34 + 2.7  2.39 + 0.81 1.49 + 0.04 1.51 + 0.02 Urine 2.44 + 1.6819.49 + 1.96  18.81 + 0.85  19.88 + 3.03 Lung 1.08 + 0.15 0.16  0.1 +0.04 0.06 + 0.01 Liver 19.9 + 0.87 5.06 + 0.89 5.3 + 0.9 3.81 + 0.92 GI13.6 + 0.8  62.7 + 2.7  68.6 + 0.4  70.1 + 1.9  Tract Heart 0.33 + 0.060.04 + 0.02 0.02 + 0.02 0.01 + 0.04

Compound 8

2 min 15 min 30 min 60 min 240 min Muscle 21.6 ± 2.08 3.97 ± 0.56 1.6 ±0.82 1.22 ± 0.28 0.22 ± 0.31 Blood 22.59 ± 1.44  1.22 ± 0.06 0.55 ± 0.190.39 ± 0.05  0.1 ± 0.02 Kidney 4.56 ± 0.29  1.9 ± 0.95  1.8 ± 0.42 1.06± 0.22 0.85 ± 0.08 Urine 0.13 ± 0.06 6.23 ± 1.23 7.18 ± 1.24 7.71 ± 0.498.55 ± 2.76 Lung 1.61 ± 0.06 0.21 ± 0.03  0.1 ± 0.04 0.08 ± 0.01 0.05 ±0.02 Liver 19.6 ± 2.16 10.0 ± 0.86 4.53 ± 0.5  2.63 ± 0.38 1.39 ± 0.29GI Tract 9.75 ± 1.2  70.9 ± 1.3  80.7 ± 2.4  85.4 ± 0.12 87.5 ± 2.8 Heart 0.63 ± 0.17 0.06 ± 0.04 n.d. 0.03 ± 0.01 0.01 ± 0.02 n.d. notdetectable.

Compound 16

2 min 30 min 60 min 240 min Muscle 32.9 + 0.97 4.63 + 0.25 1.51 + 0.530.55 + 0.08 Blood 17.8 ± 1.78 2.20 ± 0.5  0.38 ± 0.14 0.04 ± 0.04 Kidney10.55 ± 1.1  3.47 ± 0.71 1.80 ± 0.65 1.06 ± 0.06 Urine 1.74 ± 1.05 48.0± 2.22 58.2 ± 8.87 58.9 ± 1.66 Lung 1.53 ± 0.12 0.31 ± 0.08 0.09 ± 0.030.04 ± 0.00 Liver 5.61 ± 0.23 5.15 ± 0.75 3.07 ± 0.47 2.01 ± 0.11 GI6.61 ± 0.43 27.3 ± 3.45 31.6 ± 6.4  36.5 ± 1.5  Tract Heart 0.44 ± 0.090.06 ± 0.01 0.01 ± 0.01 0.00

Compound 17

2 min 15 min 30 min 60 min 240 min Muscle 25.9 + 1.8  15.6 + 1.05 8.51 +0.93 4.31 + 0.95 0.81 + 0.26 Blood 18.5 + 1.06 9.46 + 0.24  5.7 + 0.422.23 + 0.15 0.09 + 0.02 Kidney 12.3 + 0.53 7.32 + 0.76  7.1 + 1.893.77 + 0.73  5.1 + 0.99 Urine 1.38 + 1.34 30.1 + 1.28 46.9 + 3.71 65.8 +3.4  66.08 + 4.98  Lung 1.69 + 0.1   0.9 + 0.16 0.54 + 0.07 0.28 + 0.02 0.1 + 0.01 Liver  4.3 + 0.06 4.25 + 0.85 2.94 + 0.22 1.79 + 0.45   1 +0.17 Spleen 0.33 + 0.07 0.17 + 0.04 0.11 + 0.03 0.07 + 0.02 0.03 + 0.02GI Tract 6.31 + 0.6  9.6 + 0.9 13.07 + 1.4  17.7 + 3.8  25.4 + 5.2 Heart 0.34 + 0.17 0.27 + 0.06 0.17 + 0.04 0.07 + 0.02 0.01

Compound 24

2 min 15 min 30 min 60 min 240 min Muscle 28.5 + 0.25 17.9 + 4.38 10.2 +1.09 3.62 + 0.9   1.1 + 0.32 Blood 18.2 + 1.39 7.11 + 1.29 3.86 + 0.62 1.6 + 0.57 0.29 + 0.06 Kidney 10.2 + 3.07 4.11 + 1.31 2.52 + 1.1  1.4 + 0.38 0.92 + 0.37 Urine 0.11 + 0.1  8.64 + 5.6  31.0 + 9.41 44.9 +7.32 49.51 + 4.62  Lung 1.99 + 0.12 0.78 + 0.16 0.39 + 0.06 0.18 + 0.060.05 + 0.02 Liver 5.38 + 0.75  6.7 + 1.21 3.67 + 0.62  1.6 + 0.29  0.3 +0.03 GI Tract 7.48 + 0.45 23.3 + 6.5  31.5 + 11.9 38.8 + 9.6  46.3 +4.4  Heart 0.73 + 0.1   0.3 + 0.08 0.13 + 0.03 0.06 + 0.03 0.02 + 0.01

Compound 25

2 min 15 min 30 min 60 min 240 min Muscle 25.7 + 2.0  8.93 + 0.38 6.43 +0.41 4.95 + 0.09  1.3 + 0.38 Blood 23.0 + 2.9   3.9 + 0.17 2.12 + 0.341.84 + 0.19 0.62 + 0.16 Kidney 5.37 + 1.3  7.13 + 0.4  5.6 + 0.1 4.24 +0.4  3.19 + 0.1  Urine 0.11 + 0.06 4.62 + 2.9  11.6 + 2.0  17.5 + 1.7 16.81 + 0.43  Lung 3.42 + 0.39 1.37 + 0.03 1.17 + 0.01 0.96 + 0.110.21 + 0.05 Liver 13.6 + 2.47 13.5 + 0.14 9.66 + 1.65 6.73 + 0.48 3.97 +0.53 GI Tract 8.53 + 0.66 45.68 + 4.1  54.7 + 2.09 55.87 + 2.5  71.3 +0.7  Heart 0.74 + 0.1  0.16 + 0.01  0.1 + 0.03 0.08 + 0.03 0.02 + 0.01

Example 28 Incorporation into Clots Induced in a Rat Model

Rat Inferior Vena Cava Model (IVC)

The rats (Male Wistar,250-350 g) were anaesthetised with 15% urethane.After laparotomy, the vena cava was isolated and freed of surroundingfat tissue. A platinum wire (1.5 cm×0.5 mm) was inserted into theinferior vena cava and 5 min post surgery 0.4 ml of ellagic acid(1.2×10⁻⁴M) was injected intravenously through the femoral veinpreviously canulated, and the clot was allowed to form. The averageweight of the clots formed in this model was around 27 mg, n=32, (5-50mg range). The compounds were injected 5 min post-induction. After 60min the animals were sacrificed and the clot removed, weighed andcounted. Other tissues e.g. blood, lung, heart, were also dissected andcounted . The uptake of tracer into the clot was determined as therelative concentration (cpm/g of clot by dose/g animal) and clot tobackground tissue.

Results

Tc-Cpd3 Tc-Cpd4 Tc-Cpd8 Tc-Cpd14 Tc-Cpd16 Tc-Cpd17 Tc-Cpd24 Tc-Cpd25 %ID/g 0.10 ± 0.02 0.18 ± 0.06 0.17 ± 0.09 0.18 ± 0.03 0.27 ± 0.06 0.32 ±0.04 0.21 ± 0.02 0.29 ± 0.09 Relative 0.37 ± 0.1  0.52 ± 0.2  0.59 ±0.31 0.54 ± 0.11 0.93 ± 0.16  1.1 ± 0.17 0.62 ± 0.1  0.8 ± 0.2 concClot/Blood 2 2.3 4.1 1.3 1.7 1.15 1.8 3.3 Clot/Lung 2.2 2.7 4.4 1.5 2.11.54 2.5 1.5 Clot/Heart 3.4 4.0 8.1 2.8 3.8 2.7 3.9 5 Clot/Liver 0.230.27 0.40 2.6 0.37 1.26 0.93 0.65

Abbreviations

BOC ^(t)butoxycarbonyl

br.s broad singlet

d chemical shift in parts per million (ppm)

DCC dicyclohexylcarbodiimide

DIPEA diisopropylethylamine

DMAP dimethylaminopyridine

DMC dimethylcasein

DMF dimethylformamide

ES electrospray

EtOAc ethyl acetate

FAB fast atom bombardment

Fmoc fluorenylmethoxycarbonyl

HBTU O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate

HPLC high performance liquid chromatography

hrs hours

Hz Hertz

J coupling constant

m multiplet

MALDI-tof matrix assisted laser desorption-ionised time of flight

meOH methanol

mins minutes

^(i)PrOH iso-propanol

Py-BOP benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate

RCP radiochemical purity

RP-HPLC reverse phase high performance liquid chromatography

s singlet

t triplet

TEA triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

Z benzyl carbamate

What is claimed is:
 1. A composition comprising a conjugate of formula(metal complexing agent)—((Y)_(m)—A—NHR)_(k), where: k is a naturalnumber; Y is the same or different at different locations within themolecule and is independently chosen from: an A group, a C₄₋₈cycloheteroalkylene group, a C₄₋₈ cycloalkylene group, a C₅₋₁₂ arylenegroup, a C₃₋₁₂ heteroarylene group, or a polyalkyleneglycol, polyacticacid or polyglycolic acid moiety, m is an integer of value 0-20, A is a3-10 atom chain of units selected from —CR_(2—), —CR═CR—, —C≡C—, —NRCO—,—CONR—, —SO₂NR—, —NRSO_(2—), or —CR₂ZCR_(2—) where Z is —CH_(2—), O, S,Se or —NR—, R is the same or different at different locations within themolecule and is independently chosen from H, C₁₋₄ alkyl, C₁₋₄ alkenyl,C₁₋₄ alkynyl, C₁₋₄ alkoxyalkyl or C₁₋₄ hydroxyalkyl, with the provisothat the complexing agent does not also have attached thereto a hypoxialocalising moiety.
 2. The composition of claim 1, wherein A is—NHCO(CH₂)₂Z(CH₂)₂—, or —SO₂NH(CH₂)₂Z(CH₂)₂—, or —(CH₂)₂Z(CH₂)₂—.
 3. Thecomposition of claim 1, wherein Z is CH₂.
 4. The composition of claim 1,wherein the at least one substituent has the formula —(Y)_(m)—A—NH₂. 5.The composition of claim 1, wherein the substituent has the formula—(Y)_(b)—Ar—SO₂NH(CH₂)₅NH₂ where b is an integer of value 0 to 19 and Aris an arylene or heteroarylene group.
 6. The composition of claim 1,wherein the complexing agent is a metal chelating agent.
 7. Thecomposition of claim 6, wherein the metal chelating agent is adiaminedioxime.
 8. A metal complex of one or more radiometal orparamagnetic metal ions with the composition of claim
 1. 9. The metalcomplex of claim 8, wherein the radiometal is ^(99m)Tc, ¹¹¹In or ⁶⁷Ga.10. The metal complex of claim 8 for use in the diagnosis or therapy ofthrombosis, embolism, atherosclerosis, inflammation or cancer.
 11. A kitfor the preparation of the metal complex of claim
 8. 12. A vesselcontaining a unit dose for human administration of the metal complex ofclaim
 8. 13. A method of preparing a composition for use in thediagnosis or therapy of thrombosis, atherosclerosis, inflammation orcancer, which method comprises bringing the metal complex of claim 8into a form suitable for human administration.